Studies of degenerative hereditary ocular conditions, including Retinitis Pigmentosa (RP) and various macular dystrophies, have resulted in a substantial elucidation of the molecular basis of these debilitating human retinal degenerations. Applying the approach of genetic linkage, x-linked RP (xlRP) genes have been localised to the short arm of the X chromosome (Ott et al. 1990). Subsequently, the gene involved in one form of xlRP was identified. Various genes involved in autosomal dominant forms of RP (adRP) have been localised. The first of these mapped to 3q, close to the gene encoding the rod photoreceptor protein rhodopsin (McWilliam et al. 1989; Dryja et al. 1990). Similarly, an adRP gene was placed on 6p close to the gene encoding the photoreceptor protein peripherin (Farrar et al. 1991a,b; Kajiwara et al. 1991). Other adRP genes have been mapped to discrete chromosomal locations; however the disease genes as yet remain uncharacterised. As in xlRP and adRP, various genes involved in autosomal recessive RP (arRP) have been localised and in some cases molecular defects characterised (Humphries et al. 1992; Farrar et al. 1993; Van Soest et al. 1994). Similarly, a number of genes involved in macular dystrophies have been mapped (Mansergh et al. 1995). Genetic linkage, together with techniques for mutational screening of candidate genes, enabled identification of causative dominant mutations in the genes encoding rhodopsin and peripherin. Globally, about 100 rhodopsin mutations have been found in patients with RP or congenital stationary night blindness. Similarly, approximately 40 mutations have been characterised in the peripherin gene in patients with RP or macular dystrophies. Knowledge of the molecular aetiology of these retinopathies has stimulated the generation of animal models and the exploration of methods of therapeutic intervention (Farrar et al. 1995; Humphries et al. 1997).
Similar to RP, osteogenesis imperfecta (OI) is an autosomal dominantly inherited human disorder whose molecular pathogenesis is extremely genetically heterogeneous. OI is often referred to as ‘brittle bone disease’, although additional symptoms including hearing loss, growth deficiency, bruising, loose joints, blue sclerae and dentinogenesis imperfecta are frequently observed (McKusick, 1972). Mutations in the genes encoding the two type I collagen chains (collagen 1A1 and 1A2) comprising the type I collagen heterodimer have been implicated in OI. Indeed hundreds of dominantly acting mutations have been identified in OI patients in these two genes, many of which are single point mutations, although a number of insertion and deletion mutations have been found (Willing et al. 1993; Zhuang et al. 1996). Similarly mutations in these genes have also been implicated in Ehlers-Danlos and Marfan syndromes (Dalgleish et al. 1986; Phillips et al. 1990; D'Alessio et al. 1991; Vasan NS et al. 1991).
Generally, gene therapies utilising viral and non-viral delivery systems have been used to treat inherited disorders, cancers and infectious diseases. However, many studies have focused on recessively inherited disorders, the rationale being that introduction and expression of the wild type gene may be sufficient to prevent/ameliorate the disease phenotype. In contrast gene therapy for dominant disorders requires suppression of the dominant disease allele. Notably many of the characterised mutations causing inherited diseases such as RP or OI are inherited in an autosomal dominant fashion. Indeed there are over 1,000 autosomal dominantly inherited disorders in man. In addition, there are many polygenic disorders due to co-inheritance of a number of genetic components which together give rise to the disease state. Effective gene therapies for dominant or polygenic diseases may be targeted to the primary defect and in this case may require suppression of the disease allele while in many cases still maintaining the function of the normal allele. This is particularly relevant where disease pathology is due to a gain of function mutation rather than due to reduced levels of wild type protein. Alternatively, suppression therapies may be targeted to secondary effects associated with the disease pathology, for example, programmed cell death (apoptosis), which has been observed in many inherited disorders.
Strategies to differentiate between normal and disease alleles and to selectively switch off the disease allele using suppression effectors such as antisense DNA/RNA, PNAs, ribozymes, or triple helix forming DNA, targeted towards the disease mutation may be difficult in many cases—frequently disease and normal alleles differ by only a single nucleotide. A further difficulty inhibiting development of gene therapies is the heterogeneous nature of some dominant disorders—many different mutations in the same gene give rise to a similar disease phenotype. Development of specific gene therapies for each of these may be prohibitive in terms of cost.
Suppression effectors have been used previously to achieve specific suppression of gene expression. Antisense DNA and RNA has been used to inhibit gene expression in many instances. Modifications, such as phosphorothioates, have been made to oligonucleotides to increase resistance to nuclease degradation, binding affinity and uptake (Cazenave et al. 1989; Sun et al. 1989; McKay et al. 1996; Wei et al. 1996). In some instances, using antisense and ribozyme suppression strategies has led to reversal of a tumor phenotype by reducing expression of a gene product or by cleaving a mutant transcript at the site of the mutation (Carter and Lemoine 1993; Lange et al. 1993; Valera et al. 1994; Dosaka-Akita et al. 1995; Feng et al. 1995; Quattrone et al. 1995; Ohta et al. 1996). For example, neoplastic reversion was obtained using a ribozyme targeted to an H-ras mutation in bladder carcinoma cells (Feng et al. 1995). Ribozymes have also been proposed as a means of both inhibiting gene expression of a mutant gene and of correcting the mutant by targeted trans-splicing (Sullenger and Cech 1994; Jones et al. 1996). Ribozymes can be designed to elicit autocatalytic cleavage of RNA targets; however, the inhibitory effect of some ribozymes may be due in part to an antisense effect due to the antisense sequences flanking the catalytic core that specify the target site (Ellis and Rodgers 1993; Jankowsky and Schwenzer 1996). Ribozyme activity may be augmented by the use of, for example, non-specific nucleic acid binding proteins or facilitator oligonucleotides (Herschlag et al. 1994; Jankowsky and Schwenzer 1996). Multitarget ribozymes (connected or shotgun) have been suggested as a means of improving efficiency of ribozymes for gene suppression (Ohkawa et al. 1993). Triple helix approaches have also been investigated for sequence-specific gene suppression—triplex forming oligonucleotides have been found in some cases to bind in a sequence-specific manner (Postel et al. 1991; Duval-Valentin et al. 1992; Hardenbol and Van Dyke 1996; Porumb et al. 1996). Similarly, peptide nucleic acids have been shown to inhibit gene expression (Hanvey et al. 1992; Knudson and Nielsen 1996; Taylor et al. 1997). Minor groove binding polyamides can bind in a sequence-specific manner to DNA targets and hence may represent useful small molecules for future suppression at the DNA level (Trauger et al. 1996). In addition, suppression has been achieved by interference at the protein level using dominant negative mutant peptides and antibodies (Herskowitz 1987; Rimsky et al. 1989; Wright et al. 1989). In some cases suppression strategies have lead to a reduction in RNA levels without a concomitant reduction in proteins, whereas in others, reductions in RNA have been mirrored by reductions in protein.